The Mackall laboratory seeks to apply the principles of immunotherapy gleaned from our basic studies to the treatment of patients with pediatric cancer. Specifically, we have focused our efforts primarily on dissecting the immunobiology of Ewing's sarcoma. This tumor is an aggressive bone and soft tissue tumor for which outcomes remain suboptimal and no significant new therapy has been introduced for over 20 years. We seeks to use immune based therapy to improve outcomes for patients with this tumor. By conducting clinical studies of immunotherapy in patients with this disease, we acquire lymphocytes which are used to explore immune reactivity directed toward Ewing's sarcoma. Furthermore, when possible, tumor cell lines are derived from primary tumor biopsies and these cells are used to study their interactions with cells of the immune systems. Four primary accomplishements have arise from this work in the last two years. #1) Initiation of a Clinical Trial of anti-TRAIL receptor antibody in pediatric sarcomas. Based upon work in our laboratory and that of others, we demonstrated that Ewing's sarcoma is readily killed following ligation of the TRAIL-R2 antibody. Clinical development of an anti-human TR2 moAb is currently underway. We therefore have received permission from Human Genome Sciences to conduct the first Phase I trial of anti-TR2 in pediatric solid tumors. This will represent a collaboration between Memorial Sloan Kettering and the University of Cincinnati. The trial will begin this fall and will likely be open for 18 mos.-2 years. It will assess the toxicity, pharmacokinetics and clinical activity of a TR2 moAb in patients with pediatric solid tumors. If it is safe and well tolerated, we hope to conduct a Phase II trial of this agent in Ewing's sarcoma in the future. #2) Identification of a tumor specific cell surface molecule--gp96 which mediates immunomodulating activity. MAb 8H9 binds a surface molecule on a nearly all sarcomas, most brain tumors and placental tissue but not normal tissues; therefore its ligand is of broad relevance to oncology and highly relevant to pediatric oncology. We used a variety of techniques to identify that 8H9 binds the ubiquitous ER resident protein gp96. Previous paradigms held that gp96 was an immune activator and a signal of danger to the immune system. However, because the data we generated inferred that gp96:immune system interactions occurred regularly within tumor and placental microenvironments, both sites of immune evasion, we sought to more carefully study the immunobiology of the interaction between surface gp96 and DCs. This work demonstrated that gp96 can serve as a tumor-specific target since it is expressed on the surface of tumor cells but not on normal tissues. Surface expression of gp96 interacts with DCs to generate a "mature" DC which expresses low levels of co-stimulatory molecules, secretes predominantly IL-10, and is unable to stimulate CD8 recall responses. Thus, in a manner recapitulating mechanisms necessary for reproduction, interactions between gp96 and DCs within the tumor microenvironment induce tolerogenic DCs that contribute to immune escape. This work has been submitted for publication and we will seek to generate a new anti-gp96 moAb which shows higher affinity and therefore may be more effective as a tumor targeting agent in vivo. #3) Based upon our previously published results demonstrating that 4-1BB based costimulation can expand Ewing's sarcoma reactive T cells from patients with Ewing's sarcoma, we are producing a master cell bank of artificial antigen presenting cells (aAPCs) expressing 4-1BBL for use in clinical trials of adoptive immunotherapy for ES and potentially other tumors. Early results demonstrate that these artificial antigen presenting cells can induce potent expansion of antigen specific CD8+ cells ex vivo which could then be used for adoptive cellular immunotherapy.